HYDRATION CHANGES LINKED TO SEQUENCE SPECIFIC DNA-PROTEIN RECOGNITION REACTIONS[unreadable] [unreadable] We have particularly focused on DNA complexes of restriction endonucleases. Beyond their unparalleled importance as tools for the analysis and recombination of DNA, restriction enzymes have provided remarkable model systems for studying numerous aspects of protein-nucleic acid interactions. These proteins combine high binding strength and extraordinary sequence specificity. [unreadable] [unreadable] Differences in sequestered water between specific and nonspecific complexes of BamHI comparison with x-ray structures. (Sidorova and Rau)[unreadable] [unreadable] Experiments measuring the difference in sequestered water between specific and nonspecific complexes of the restriction endonuclease BamHI using the osmotic stress technique and a novel self-cleavage assay have been completed and published. Using the osmotic stress technique together with a self-cleavage assay developed by us we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes (such as betaine glycine, triethylene glycol, sucrose, et. cetera) used to set water activity. The observed osmotic dependence indicates that the nonspecific BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity indicates that these waters are sterically inaccessible to solutes. This result is in close agreement with difference in structures determined by x-ray crystallography.[unreadable] We demonstrated additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent. For example, the absolute specific binding constant of BamHI shows a weak dependence on osmotic pressure of the disaccharide sucrose translating into release of only about 46 water molecules in forming the complex. In contrast, tetrasaccharide stachyose has a very dramatic effect on specific binding corresponding to the release of 470 water molecules. Such a result is expected for reactions resulting in a large change in a surface exposed area. [unreadable] We also showed that precautions should be taken to ensure structurally meaningful results. A variety of solutes of different sizes and chemical natures should be used to probe differences in hydration. For example, solutes as methanol and glycerol are small enough to penetrate narrow cavities resulting in significant underestimation of the actual number of sequestered waters. [unreadable] [unreadable] EcoRV binding to specific and nonspecific DNA sequences (Sidorova and Rau).[unreadable] [unreadable] The novel techniques we have developed utilizing osmotic stress are broadly applicable to measuring DNA-protein interactions. We have become interested in another type II restriction enzyme, EcoRV. Usually restriction endonucleases are capable to distinguish between cognate and nonspecific DNA sequences quite efficiently in the absence of divalent cofactor that is required for cleavage. For example, EcoRI restriction endonuclease binds its specific site on DNA with binding constant that is at least 10,000 -fold higher than the nonspecific binding constant in the absence of divalent cations, there is an additional increase of binding specificity coupled to the presence of Mg2+ or Ca2+. At the same time, there are many conflicting results in literature regarding ability of the EcoRV to distinguish between specific and nonspecific DNA sequences in the absence of divalent ions. These studies were done utilizing standard gel mobility shift and filter binding assays as well as fluorescent techniques. One group only has demonstrated significant specificity. The majority of researchers do not see meaningful preferential binding, typically less than a 10-fold difference between the recognition sequence and nonspecific DNA in the absence of divalent metal ions. The x-ray structures for specific and non-cognate DNA-EcoRV complexes are, however, substantially different suggesting it is probable that EcoRV specific and nonspecific binding free energies also differ substantially. We have applied our self-cleavage assay to measure solution binding. This technique does not have the limitations of more commonly used assays as gel mobility shift, filter binding, and anisotropy of fluorescently labeled complexes. Our preliminary results are promising and indicate significant EcoRV binding specificity in the absence of divalent ions. We have also uncovered an unusual slow transition between specific binding modes that may account for the discrepancies seen in the literature. We do observe a strong dependence of the relative binding constant of EcoRV on osmotic pressure as would be expected from the x-ray structures.